1. Technical Field
The present invention is related to a synthetic blood platelet suspension medium. More particularly, the present invention is related to a synthetic preservation medium for platelets which (1) is free of blood plasma and proteins, (2) extends platelet shelf life and improves the quality of platelet concentrates stored for transfusion and (3) is free of organic compounds other than dextrose and citrate.
2. State of the Art
Blood is composed of two major portions. These portions can be recognized when a specimen of blood is taken and clotting is prevented. That portion of the blood which settles to the bottom of the vessel holding the specimen is termed the "formed elements". The formed elements comprise red blood cells and other particulate components such as white blood cells and platelets which are also known as thrombocytes. The formed elements are characteristically 40 to 50 percent of the bulk of normal human blood. The cloudy liquid which does not settle in a blood specimen is the portion of the blood known as plasma. Plasma is primarily water, but contains inorganic and organic substances as well as dissolved gases and miscellaneous foreign substances. The inorganic substances contained in blood plasma are primarily electrolytes. The most significant of these electrolytes are presented in Table 1.
TABLE 1 ______________________________________ Sodium 142.0 mEq/l Potassium 4.3 mEq/l Calcium 5.0 mEq/l Magnesium 3.4 mEq/l Chloride 104.0 mEq/l Bicarbonate 27.0 mEq/l Phosphate 2.3 mEq/l Sulfate 0.6 mEq/l ______________________________________
The most significant organic substances found in the plasma are lactic acid, urea, amino acids, creatinine, glucose, hormones, proteins, albumins, and globulins.
Modern medicine has been developing solutions that are added to blood in vivo and/or mixed with blood in vitro. Products that are used for adding to blood in vivo are primarily used for intravenous feeding, pharmaceutical vehicles, and/or electrolyte replacement in patients who are bedfast. These solutions are primarily comprised of water that contains dextrose and, optionally, electrolytes. Dextrose is typically present in these solutions in about a 5 percent concentration and provides a nutrient for blood cells or tissue cells. The electrolytes contained in these solutions vary widely. The solutions that contain electrolytes that most closely resemble blood plasma contain a plurality of the electrolytes presented in Table 1. A specific example of a dextrose and electrolyte solution suitable for in vivo addition in blood is Locke-Ringer's solution. The formula for Locke-Ringer's solution is presented in Table 2.
TABLE 2 ______________________________________ Reagent Sodium Chloride 9.0 Gm Reagent Potassium Chloride 0.42 Gm Reagent Calcium Chloride 0.24 Gm Reagent Magnesium Chloride 0.2 Gm Sodium Bicarbonate 0.5 Gm Dextrose 0.5 Gm Water, recently distilled from a 1000 ml hard glass flask, in a sufficient quantity, to make ______________________________________
Other solutions suitable for the addition of blood in vivo can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, 14th Edition (1970), pages 815 to 847.
Solutions that are added to blood in vitro are principally concerned with preserving either whole blood or separated components of the blood such as red blood cells, white blood cells, or mixtures of various substances. When platelets are included in a component of blood that is to be collected and preserved in vitro an anticoagulant is added. The most frequently used anticoagulant added to collected whole blood is known as "acid-citrate-dextrose" or "ACD". This anticoaulant solution contains (1) citric acid and sodium citrate in concentrations sufficient to produce an optimum physiological pH and (2) dextrose in concentrations sufficient for long term preservation of red blood cells. A solution that has been found desirable to preserve both whole blood and fractions of whole blood is known as "anticoagulant citrate-phosphate-dextrose solution" or "CPD". The components of anticoagulant citrate-phosphate-dextrose solution are presented in Table 3.
TABLE 3 ______________________________________ Citric Acid (anhydrous) 3.0 Gm Sodium Citrate (dihydrate) 26.3 Gm Sodium Biphosphate (monohydrate; 2.22 Gm NaH.sub.2 PO.sub.4 H.sub.2 O) Dextrose 25.5 Gm Water for Injection, in a 1000 ml sufficient quantity to make ______________________________________
Specific elements of the particulate component of blood can be separated and preserved for later transfusion. Traditional processes can be used to collect and preserve white blood cells and platelets together. More modern processes allow platelets to be separated, stored, and reinfused into recipients suffering from platelet deficiency. Rapid deterioration of these elements occurs after separation and storage by these processes. It is hypothesized that deterioration in platelet quality during storage is due to the activation of plasma clotting factors released during storage.
The storage of separated platelets or "platelet concentrates" that are intended for transfusion is typically conducted by one of three processes. These processes involve platelet suspension in gelatin followed by chilling of the suspension, freezing or freezing and lyophilizing platelets, and liquid storage of platelets. These processes are generally described in Hematology, Williams et al., Second Edition, McGraw-Hill Book Company (1977), pages 1553-61, herein incorporated by reference.
One of the oldest techniques known for the storage of platelets is disclosed in U.S. Pat. No. 2,786,014 to Tullis. The platelets are suspended in 40 milliliters of water with between 0.3 to 1.2 grams of gelatin with sodium acetate and sodium chloride. The suspension is stored at 4.degree. C. This process has generally been discontinued because chilling or low temperatures cause morphological changes that alter the normal discoid shape of platelets into a spherical shape. This distortion of the platelet shape at temperatures below 15.degree. C. becomes permanent after a few hours.
The technique of platelet storage by freezing has been known for years, but has does not have wide clinical application. In this process platelets are suspended in a 5 to 10 percent glycerol or glycerol-glucose solution and frozen. The platelets can remain frozen for days or months before thawing and reinfusing. The recovery of viable platelets after freezing and thawing is only about 30 percent. This recovery of viable platelets is about one-half of that recovered by processes that separate fresh platelets and do not involve freezing. This loss of viable platelets decreases the desirability of the freezing process.
The most promising processes for platelet storage involve liquid storage of platelets in temperatures, that do not produce morphological damage to the stored platelets, such as temperatures of about 22.degree. C. The solutions for liquid storage of platelets generally include one or more of the electrolytes listed in Table 1, dextrose or glucose, an anticoagulant, and one or more additives. These solutions typically preserve platelets for about 24 to about 72 hours. Some additives can only be used for experimental purposes because the additives fail to meet safety or regulatory requirements or, as with many organic and especially proteinaceous compounds, can sensitize the recipient and cause allergic reactions upon repeated exposure to the compounds.
Platelet concentrates for most clinical purposes are currently prepared from collected units of citrate-phosphate-dextrose anticoagulated whole blood and stored in approximately 50 to 60 milliliters of the anticoagulated plasma or "CPD-plasma". The CPD-plasma is infused together with the platelets into patients in need of platelet transfusions. In that this process requires the use of plasma for the storage of the platelets, that plasma is not available for other purposes in the treatment of patients. It is, therefore, desirable to store or suspend viable platelets in a plasma-free medium so as to not detract from the amount of collected plasma available for transfusion into patients. Additionally, the plasma used to suspend platelets can cause an allergic reaction to occur in a patient after a transfusion because of a blood type or "ABO" incompatibility between a donor and a recipient of the transfused platelets.
Other studies describing preservation media for platelets were published by Baldini et al., Blood 15: 909 (1960) and Bagdasarov et al., Blood 16: 1667 (1960). The preservation medium described by Baldini contained inosine and adenine in addition to glucose, plasma, and a phosphate buffer. Baldini et al. demonstrated that about 40% of the platelets can remain viable using radiolabeling after six days of storageat 4.degree. C. However, the infused platlets disappeared from the circulation in the undesirably short period of time of less than 20 hours. The preservation media described by Bagdasarov et al. is a salt solution containing glucose and EDTA. The Bagdasarov et al. solution also requires storage at 4.degree. C. The in vitro results of this disclosure had undesirably low platelet viability. Storage of the platelets at 4.degree. C. and the use of EDTA decrease the function and viability of the platelets.
U.S. Pat. No. 3,629,071 to Sekhar discloses preservation solutions for platelets containing glucose, magnesium chloride, and prostaglandins. The suspensions of this patent preserve the hemostatic function of the platelets. This function was demonstrated by incubating rat platelet rich plasma in the presence of PGE for 48 hours at 4.degree. C. and performing aggregation experiments. The suitability of using these solutions for storage of human platelet concentrates for transfusion and the in vivo viability of platelets stored in these solutions is not documented. The use of additives such as prostaglandins is for experimental purposes and to date is not approved by the U.S. Food and Drug Administration for use in substances for infusion into humans.
U.S. Pat. No. 4,390,619 to Harmening-Pittiglio discloses a platelet storage medium using an ion-exchange resin. The improvement presented by this invention consists of a water insoluble polymer containing releasable phosphate or bicarbonate ions. These ions supply a physiologically acceptable, sustained release of buffer in the medium. The sustained release of buffer maintains both pH and ATP levels that are equal to at least 60% of the level found in freshly prepared platelets. The medium can store platelets for a period of at least seven days at 22.degree. C. A substantial and undesirable loss of platelets occurs with this medium during storage in the resin bags. For example, at day 7 and with 1 gram of resin the reported platelet count is, respectively, 43 percent of the initial platelet count in Fenwal bags and 61 percent of the initial platelet count in Cutter bags. There is no documentation in this patent of in vivo studies demonstrating platelet viability upon infusion.
Studies using a plasma-free medium for storage of platelet concentrates have been described in an abstract by Adams et al., "Abstracts of the 18th Congress of the International Society of Blood Transfusion Munchen", July, 1984, page 124. The composition of this medium is not disclosed, but platelets can be stored in this medium for up to five days and produce in vitro results similar to that obtained by storing platelets in plasma. No in vivo studies are reported in this document.
U.S. Pat. No. 4,447,415 to Rock et al. discloses a liquid storage medium for platelets that is plasma-free. The medium of this invention uses one or more additives in conjunction with a saline and anticoagulant, dextrose-containing solution that is desirably a form of CPD-Tyrode's solution. The additives disclosed as being suitable for use with this invention include (1) reversible inhibitors that are organic compounds such as indomethacin, quinacrine, or vitamin E and (2) substances or raise cyclic adenosine monophosphate levels such as prostaglandins E.sub.1, D.sub.2, or I.sub.2. As stated above many of these additives fail to meet safety and regulatory requirements required for substances for infusion into humans and are, therefore, only suitable for experimental use or only for in vitro use. Other additives disclosed as suitable for use with this invention include (1) nutrients such as fructose and other sugars, adenine, or acetyl CoA and (2) buffers such as phosphate and certain amino acids. The organic compounds or additives identified as nutrients do not eliminate the requirement for the presence of dextrose in the medium and do not satisfy the nutrient requirement for the platelets for periods of storage time extending beyond about 5 days. The additives identified as buffers cannot maintain a balanced pH during extended platelet storage periods beyond about 5 days. These buffers cannot adequately buffer the amount of lactic acid produced by viable, suspended platelets as a by-product from the consumption of dextrose that occurs when the platelets are stored at temperatures of at least about 22.degree. C.
The industry is lacking a platelet storage medium that is free of plasma and organic compounds, other than dextrose and an anticoagulant such as citric acid, and preserves platelets without chilling or in temperatures of at least about 22.degree. C. for storage periods of more than 7 days with minimal loss of viability and without the use of additives that are either unsafe or unapproved for in vivo human use.